Pseudonocardia autotrophica is one of actinomycetes and is known to have an ability to convert the vitamin D group such as vitamin D3 from an inactive form to an active form (K. Takeda, J. Ferment. Bioeng., 78(5), 380-382 (1994); Non Patent Document 1).
Vitamin D3 synthesized in biological synthesis systems is usually in an inactive form and shows little physiological activities without further treatments. The inactive form of vitamin D3 is hydroxylated at positions 25 and 1α in the liver and kidney, respectively, and is converted into the active form of vitamin D3 (1α,25-dihydroxyvitamin D3) which shows various physiological activities. Therefore, the hydroxylation reaction from the inactive form to the active form is a particularly important step in expression of a function of vitamin D3.
The active form of vitamin D3 is known to promote absorption of calcium into the body and deposition of calcium to bone tissues, and deficiency of vitamin D3 causes a variety of diseases due to an abnormality of calcium metabolism, such as osteoporosis. Further, in recent years, involvement of the active form of vitamin D3 in cellular differentiation induction and immune regulation has attracted attention. Therefore, the active form of vitamin D3 can be used as a drug for improving or treating a disease caused by the abnormality of calcium metabolism, cellular differentiation, immune regulation, or the like.
As mentioned above, the active form of vitamin D3 can be used as a drug for treating a variety of diseases, but in the case where the active form of vitamin D3 is industrially produced, there are problems such as complex production steps and low yield in chemical synthesis. Therefore, establishment of a more efficient method of producing the active form of vitamin D3 has been desired.
In recent years, an ischemic heart disease caused by coronary arteriosclerosis is increasing in accordance with aging of population and westernization of diets. The incidence rate of the ischemic heart disease is known to increase in the case where a serum cholesterol value exceeds a certain level (W. B. Kannel, Ann. Inntern. Med., 74, 1 (1971); Non Patent Document 2). Cholesterol present in the body includes cholesterol absorbed from a diet and cholesterol biosynthesized in a living body. In the case of humans, it is reported that the amount of cholesterol biosynthesized is 3 to 4 times larger than that of cholesterol absorbed from a diet (J. M. Dietschy, N. Engl. J. Med., 282, 1179 (1970); Non Patent Document 3). Therefore, it is expected that suppression of biosynthesis of cholesterol lowers serum cholesterol value to thereby obtain preventing and treating effects on ischemic heart diseases.
As inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is a key enzyme of cholesterol biosynthesis, compactin and pravastatin obtained by hydroxylation of compactin at position 6β have been discovered (JP 61-13699 B (U.S. Pat. No. 4,346,227, etc.) (Patent Document 1), U.S. Pat. No. 4,346,227 (Patent Document 2), U.S. Pat. No. 4,410,629 (Patent Document 3), and U.S. Pat. No. 4,448,979 (Patent Document 4)). Pravastatin exhibits an excellent cholesterol biosynthesis inhibitory activity and organ-selective inhibitory activity and hence is used as an anti-hyperlipidemia agent for treating or preventing the ischemic heart disease such as arteriosclerosis.
As methods of synthesizing pravastatin, microbiological methods each including converting compactin used as a raw material into pravastatin by hydroxylation of compactin at position 6β are known (JP 62-54476 B (U.S. Pat. No. 4,346,227, etc.) (Patent Document 5), U.S. Pat. No. 4,346,227 (Patent Document 2), U.S. Pat. No. 4,410,629 (Patent Document 3), U.S. Pat. No. 4,448,979 (Patent Document 4), and U.S. Pat. No. 5,179,013 (Patent Document 6)). However, the microbiological methods are insufficient in terms of pravastatin production ability and production efficiency, and hence establishment of a more efficient method of producing pravastatin has been desired.
The reaction from compactin into pravastatin is hydroxylation as in the case of the reaction from vitamin D3 into the active form of vitamin D3, and the use of Pseudonocardia autotrophica to be used in industrial production of the active form of vitamin D3 (K. Takeda, J. Ferment. Bioeng., 78(5), 380-382 (1994); Non Patent Document 1) has been expected to provide an efficient pravastatin production system. However, an expression vector for Pseudonocardia autotrophica serving as a host has not been reported.